title: Reliable expression and purification of highly insoluble transmembrane domains.
abstract: A general procedure for the reliable preparation of insoluble transmembrane domains has been developed. Improved expression schemes were developed by expressing the transmembrane domains of caveolin proteins 1, 2, and 3 as a fusion to the Trp leader protein. This construct readily formed inclusion bodies during overexpression, allowing high levels of protein to be achieved. Cleavage of the transmembrane domain away from the Trp leader carrier protein was performed with cyanogen bromide. The transmembrane domains were then purified using reverse-phase high-performance liquid chromatography with a C4 column and were eluted with a mixture of 1-butanol and acetic acid. Using this method, the 39-42 amino acid transmembrane domains from caveolin proteins 1, 2, and 3 were successfully purified to homogeneity. Further verification of this method was successfully done with Rfbp(18-51), another insoluble transmembrane domain.
authors.affiliation: Department of Chemistry, Lehigh University, Bethlehem, PA 18015, USA.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Diefenderfer
authors.forename: Jinwoo
authors.initials: J
authors.lastname: Lee
authors.forename: Scott
authors.initials: S
authors.lastname: Mlyanarski
authors.forename: Ying
authors.initials: Y
authors.lastname: Guo
authors.forename: Kerney Jebrell
authors.initials: KJ
authors.lastname: Glover
mesh_terms: D000595:Amino Acid Sequence; D022461:Caveolins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002479:Inclusion Bodies / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012995:Solubility
chemical_list: D022461:Caveolins